“Sometimes young researchers haven’t contemplated all the possibilities of how to use and apply an instrument; and don’t realize there are many different ways to utilize familiar tools in order to obtain new, in some cases better, information,” McCaffery said. “Our desire is always to approach a problem from many disparate perspectives to generate convergent data that corroborates each particular assay. Hopefully, results from each individual assay, allows the scientist to arrive at a convergent perspective that yields confidence in the results and conclusions.”

One of the easiest ways to obtain different microscopy data and improve corroboration among assays is simply to change the contrast mechanism.

“The most common contrast mechanisms used to image something are optical contrast (transparent versus opaque), polarization, and fluorescence,” said Wilson. “But there are many different ways you can manipulate how light interacts with the specimen and what you detect out of an objective.”

For example, ultrafast laser sources have made nonlinear optical forms of contrast an exciting new tool. Techniques like two-photon excited fluorescence and second harmonic generation (both available in the IIC) produce excellent spectral and structural information about samples because a smaller effective photon volume is excited. Wilson explained it like this: “Imagine turning your stereo all the way up and hearing the sound distorted. That distortion is created by the higher order acoustic harmonics from your stereo. The same happens with intense laser light resulting in new “colors” being generated from the object irradiated. The cool thing is that the different non-linear processes are often sensitive to different physical proper- ties or structural features, offering complementary information about your sample.”

In some cases, getting more detailed information simply requires looking at the right color range. The two-photon fluorescence and second harmonic signals appear at different wavelengths. If you excite a sample with enough energy to generate third order harmonics, that signal is detected at an even bluer wavelength, Wilson said. “With third harmonic generation, you only get signals from the interface of structures with no interference from anything else. This means you can simultaneously image fluorescence, polar order, and interface dynamics just by popping in a few filters and beam splitters,” he said.

“Over the past ten or so years, physicists and engineers focused on advanced microscopy, have produced better and more advanced laser and optical technologies, generating techniques that many researchers in the biological and biomedical sciences might not know exist,” Wilson said. “There also are a lot of applied physicists who are developing and using these new technologies who don’t know what an interesting sample is. We hope to help bridge this gap, becoming a place where these collaborative synergies can flourish.”

Sample preparation is another area where the center can help researchers. “Cell fractionation, for example, which is the breaking down of whole cells and separating them into their individual components, when combined with biochemical techniques and microscopy, can often allow researchers to pose more precise questions and to better analyze a biological problem,” McCaffery said.

“It is common for someone to come in and want to use a particular instrument or technique they read about in a paper,” McCaffery said. When that happens, McCaffery and Wilson are likely to give researchers “homework.”

“It’s important to remember that the goal is not to make a pretty picture,” Wilson said. “The goal is to answer a question, so sometimes we have to ask them, ‘What is your research question?’” An enviable set of microscopy tools combined with a team that brings years of training and experience from a variety of disciplines sets Johns Hopkins Integrated Imaging Center apart from the microscope on the individual researcher’s lab bench, as well as from facilities nationwide. Wherever possible, McCaffery said, IIC staff tries to be engaged in all of the research that is carried out in the center. “Simply, our involvement leads to better results and better science,” McCaffery added.

Researchers confirm this successful combination.

“The facilities at the IIC have allowed us to obtain critical information about the internal structure of our peptide nanomaterials that would have remained unknown without careful electron and fluorescence microscopy,” said J.D. Tovar, assistant professor of Chemistry. “Equally important, the scientific IIC staff members were vital participants making sure collaborative experiments were done meaningfully and students were trained competently. Our collaboration with Dr. Wilson has given some nice insights and at the same time has posed many more questions for future research.”

Praise like that for the IIC is always nice to hear, staff members say, but they emphasize that the services and tools they provide are just part of the job. “Part of being a scientist is learning not only how to gather information from a wide variety of tools but also understanding how to pose clear questions that lead to the right tools, in a nutshell, how to not waste time. If we can help you do that, then we have achieved our goal,” Wilson said.
This story originally appeared in Johns Hopkins Nano-Bio Magazine.

To read more about IIC’s facilities and services, go here.

Story by Mary Spiro

Photos by Mary Spiro and Marty Katz